A novel method to enhance acoustic droplet bioprinting quality through the analysis of parametric influence mechanisms
Three-dimensional bioprinting ushers a transformative change in tissue engineering, providing unparalleled opportunities for regenerative medicine by precisely fabricating intricate, biomimetic tissues. To achieve true organ-level in vitro tissue construction, various advanced bioprinting technologies have been developed. Among these, acoustic droplet bioprinting technology, owing to its excellent biocompatibility and multi-sample handling capabilities, offers an efficient, non-contact liquid-handling approach for tissue engineering applications. To meet the printing structure’s geometric precision requirements, meticulous control of printing parameters is essential. However, the selection of acoustic droplet printing parameters still depends heavily on empirical values, which often leads to print outcomes that fall short of optimal standards. In this paper, a parameterized droplet dispensing method for multi-sample droplet excitation was established. This method introduces a unified scaling parameter based on the product of surface tension and viscosity, integrating acoustic stress and fluid response into a single dimensionless quantity, thereby enabling precise adjustment of droplet velocity. The relative error between the initial velocity measured using this method and the preset velocity was less than 6.7%. Next, we analyzed the effects of droplet overlap distance and the Weber and Ohnesorge numbers on printed line-width consistency. By employing optimized printing parameters, we achieved controllable printing of patterned hydrogel meshes suitable for cell culture. The results demonstrated that the lengths and widths of the nine sub-meshes exhibited high consistency. These advances move acoustic droplet bioprinting from an experience-driven process toward a more systematic, predictive, and reproducible parameter-optimization strategy.

- Lechler RI, Sykes M, Thomson AW, Turka LA. Organ transplantation—how much of the promise has been realized? Nat Med. 2005;11(6):605-613. doi: 10.1038/nm1251
- Giwa S, Lewis JK, Alvarez L, et al. The promise of organ and tissue preservation to transform medicine. Nat Biotechnol. 2017;35(6):530-542. doi: 10.1038/nbt.3889
- Berthiaume F, Maguire TJ, Yarmush ML. Tissue engineering and regenerative medicine: history, progress, and challenges. Annu Rev Chem Biomol Eng. 2011;2(1):403-430. doi: 10.1146/annurev-chembioeng-061010-114257
- Van Blitterswijk C, De Boer J. Tissue Engineering. Cambridge, MA, USA: Academic Press; 2022. doi: 10.1016/C2020-0-01481-7
- Paxton N, Smolan W, Böck T, Melchels F, Groll J, Jungst T. Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability. Biofabrication. 2017;9(4):044107. doi: 10.1088/1758-5090/aa8dd
- Ozbolat IT, Hospodiuk M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials. 2016;76(37):321-343. doi: 10.1016/j.biomaterials.2015.10.076
- Li X, Liu B, Pei B, et al. Inkjet bioprinting of biomaterials. Chem Rev. 2020;120(19):10793-10833. doi: 10.1021/acs.chemrev.0c00008
- Angelopoulos I, Allenby MC, Lim M, Zamorano M. Engineering inkjet bioprinting processes toward translational therapies. Biotechnol Bioeng. 2020;117(1):272-284. doi: 10.1002/bit.27176
- Guillotin B, Souquet A, Catros S, et al. Laser assisted bioprinting of engineered tissue with high cell density and microscale organization. Biomaterials. 2010;31(28):7250-7256. doi: 10.1016/j.biomaterials.2010.05.055
- Ventura RD. An overview of laser-assisted bioprinting (LAB) in tissue engineering applications. Med Lasers Eng Basic Res Clin Appl. 2021;10(2):76-81. doi: 10.25289/ML.2021.10.2.76
- Melchels FP, Feijen J, Grijpma DW. A review on stereolithography and its applications in biomedical engineering. Biomaterials. 2010;31(24):6121-6130. doi: 10.1016/j.biomaterials.2010.04.050
- Skoog SA, Goering PL, Narayan RJ. Stereolithography in tissue engineering. J Mater Sci Mater Med. 2014;25(3):845-856. doi: 10.1007/s10856-013-5107-y
- Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014;32(8):773-785. doi: org/10.1038/nbt.2958
- Krainer S, Smit C, Hirn U. The effect of viscosity and surface tension on inkjet printed picoliter dots. RSC Adv. 2019;9(54):31708-31719. doi: 10.1039/c9ra04993b
- Castro JO, Ramesan S, Rezk AR, Yeo LY. Continuous tuneable droplet ejection via pulsed surface acoustic wave jetting. Soft Matter. 2018;14(28):5721-5727. doi: 10.1039/c7sm02534c
- Guo Q, Su X, Zhang X, Shao M, Yu H, Li D. A review on acoustic droplet ejection technology and system. Soft Matter. 2021;17(11):3010-3021. doi: 10.1039/d0sm02193h
- Derakhshanfar S, Mbeleck R, Xu K, Zhang X, Zhong W, Xing M. 3D bioprinting for biomedical devices and tissue engineering: a review of recent trends and advances. Bioact Mater. 2018;3(2):144-156. doi: 10.1016/j.bioactmat.2017.11.008
- Hadimioglu B, Stearns R, Ellson R. Moving liquids with sound: the physics of acoustic droplet ejection for robust laboratory automation in life sciences. J Lab Autom. 2016;21(1):4-18. doi: 10.1177/2211068215615096
- Lagus TP, Edd JF. High-throughput co-encapsulation of self-ordered cell trains: cell pair interactions in microdroplets. RSC Adv. 2013;3(43):20512-20522. doi: 10.1039/C3RA43624A
- Jentsch S, Nasehi R, Kuckelkorn C, Gundert B, Aveic S, Fischer H. Multiscale 3D bioprinting by nozzle‐free acoustic droplet ejection. Small Methods. 2021;5(6):2000971. doi: 10.1002/smtd.202000971
- Xia Y, Chen H, Li J, et al. Acoustic droplet-assisted superhydrophilic–superhydrophobic microarray platform for high-throughput screening of patient-derived tumor spheroids. ACS Appl Mater Interfaces. 2021;13(20):23489-23501. doi: 10.1021/acsami.1c06655
- Chen K, Jiang E, Wei X, et al. The acoustic droplet printing of functional tumor microenvironments. Lab Chip. 2021;21(8):1604-1612. doi: 10.1039/d1lc00003a
- Gong Z, Huang L, Tang X, et al. Acoustic droplet printing tumor organoids for modeling bladder tumor immune microenvironment within a week. Adv Healthc Mater. 2021;10(22):2101312. doi: 10.1002/adhm.202101312
- Fang Y, Frampton JP, Raghavan S, et al. Rapid generation of multiplexed cell cocultures using acoustic droplet ejection followed by aqueous two-phase exclusion patterning. Tissue Eng Part C Methods. 2012;18(9):647-657. doi: 10.1089/ten.TEC.2011.0709
- Guo Q, Shao M, Su X, Zhang X, Yu H, Li D. Controllable droplet ejection of multiple reagents through focused acoustic beams. Langmuir. 2021;37(51):14805-14812. doi: 10.1021/acs.langmuir.1c02450
- Gudapati H, Dey M, Ozbolat I. A comprehensive review on droplet-based bioprinting: past, present and future. Biomaterials. 2016;102:20-42. doi: 10.1016/j.biomaterials.2016.06.012
- Gong Y, Bi Z, Bian X, et al. Study on linear bio-structure print process based on alginate bio-ink in 3D bio-fabrication. Bio Des Manuf. 2020;3(2):109-121. doi: 10.1007/s42242-020-00065-9
- Xiao S, Zhao T, Wang J, et al. Gelatin methacrylate (GelMA)-based hydrogels for cell transplantation: an effective strategy for tissue engineering. Stem Cell Rev Rep. 2019;15(5):664-679. doi: 10.1007/s12015-019-09893-4
- Mamidi N, Ijadi F, Norahan MH. Leveraging the recent advancements in GelMA scaffolds for bone tissue engineering: an assessment of challenges and opportunities. Biomacromolecules. 2023;25(4):2075-2113. doi: 10.1021/acs.biomac.3c00279
- Lemmo AV, Fisher JT, Geysen HM, Rose DJ. Characterization of an inkjet chemical microdispenser for combinatorial library synthesis. Anal Chem. 1997;69(4):543-551. doi: 10.1021/ac960808v
- Gao H, Luo X, Cui D, et al. A study of film thickness and hydrodynamic entrance length in liquid laminar film flow along a vertical tube. AIChE J. 2018;64(6):2078-2088. doi: 10.1002/aic.16081
- Ji Y, Yang QZ, Huang GY, et al. Improved resolution and fidelity of droplet-based bioprinting by upward ejection. ACS Biomater Sci Eng. 2019;5(8):4112-4121. doi: 10.1021/acsbiomaterials.9b00400
- Qian J, Law CK. Regimes of coalescence and separation in droplet collision. J Fluid Mech. 1997;331:59-80. doi: 10.1017/S0022112096003722
- Brazier-Smith P, Jennings S, Latham J. The interaction of falling water drops: coalescence. Proc R Soc Lond A Math Phys Sci. 1972;326(1566):393-408. doi: 10.1098/rspa.1972.0016
- Cui Z, Feng Y, Liu F, Jiang L, Yue J. 3D bioprinting of living materials for structure-dependent production of hyaluronic acid. ACS Macro Lett. 2022;11(4):452-459. doi: 10.1021/acsmacrolett.2c00037
